CN103555843A - Microsatellite colorectal cancer instability amplification system and detection kit thereof - Google Patents
Microsatellite colorectal cancer instability amplification system and detection kit thereof Download PDFInfo
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Abstract
The invention relates to a microsatellite colorectal cancer instability amplification system and a detection kit thereof. The amplification system disclosed by the invention comprises a general-specific chimeric primer pair and a mixture of general primers, wherein the general-specific chimeric primer pair respectively aims at microsatellite sites BAT25, BAT26, D2S123, D5S346 and D17S250; the general primer is FluD5-Up; respective positive primer 5' ends in the general-specific chimeric primer pair are provided with a sequence of the FluD5-Up; the FluD5-Up is a fluorescent-labeled primer. The amplification system and the detection kit thereof which are disclosed by the invention overcome the shortcoming that different primers require for multiple fluorescent labels; five microsatellite gene sites are detected in the same PCR (polymerase chain reaction) system, so that a result is intuitive and reliable; the time can be saved, and the efficiency is high; MSI (medium-scale integration) gene sites can be effectively detected and analyzed.
Description
Technical field
The present invention relates to a kind of pcr amplification system, and the testing product that uses this amplification system, biological technical field belonged to.
Background technology
Colorectal cancer at home and abroad sickness rate all raises gradually, is one of common cancer of serious harm human health, and research finds that genomic unstable accounts for consequence in the pathogenesis of colorectal cancer.Genomic unstable comprises chromosome instability (chromosomal instability, CI) and microsatellite instability (microsatellite instability, MSI).Incidence study for colorectal cancer in the past shows, chromosome instability fixed (chromo some instability) is the major cause of Colorectal Cancer, and chromosome instability refers to that the increase of whole karyomit(e) quantity or minimizing speed accelerates.And another important mechanisms that microsatellite instability is Colorectal Cancer is found in research in recent years.
Micro-satellite claims again STR (Short tandem repeats, STRs), simple repetitive sequence (Simple sequences repeats, SSRs), by 1～6 Nucleotide, formed, have height polymorphism, be extensively present in protokaryon and eukaryotic gene group, particularly with dinucleotides tumor-necrosis factor glycoproteins, (CA/GT) n is the most common, n is multiplicity, is generally 15～60 times.Micro-satellite is from the structural analysis of gene, and tumor-necrosis factor glycoproteins is positioned at the junction region of promotor, gene coding region, intron region or intron and exon.The conjugated protein of micro-satellite energy autospecific or direct coding protein, the formation of the folding and telomere that may participate in chromatid having etc.Micro-satellite pass changes DNA structure or by being combined and bringing into play gene regulating effect with specific protein, is the high molecule marker of new, the polymorphic information content of a class.
Microsatellite instability refers to increase or the loss of the simple repeated sequence causing due to misreplication (replication error, RER).It occurs is mainly to participate in the gene function defect of pairing errors repair and produce a kind of deficient protein matter, thereby can not normally correct and copy mistake, thereby cause the change of microsatellite DNA, make it can not normally bring into play regulating and controlling effect, cause cell proliferation and prosoplasia, inspire malignant tumour and form.According to the literature, the tumour such as MSI and colorectal cancer, cancer of the stomach, carcinoma of endometrium occurs closely related.In about 15% colorectal cancer, have MSI phenomenon, wherein typical hereditary nonpolyposis colorectal cancer (hereditarynon-polyposis colorectal cancer, HNPCC) patient more than 90% is MSI knurl.MSI detects that it is significant to colorectal cancer patients.Compare with the colorectal cancer without MSI, its prognosis of colorectal cancer that carries MSI is better, and the two drug reaction is also different.5 microsatellite locus of Bethesda guiding outlines exemplary application in 1997 carry out the detection of colorectal cancer, i.e. single base repetitive sequence BAT-25 and BAT-26, double alkali yl tumor-necrosis factor glycoproteins D2S123, D5S346 and D17S250, and MSI has been made to definition.The normal DNA of comparison of tumor and coupling, in 5 sites, there are more than 2 or 2 sites to have tumor-necrosis factor glycoproteins length variations person for height MSI (highfrequency MSI, MSI-H), if only having 1 site to have length variations person is low MSI (lowfrequency MSI, MSI-L), variation without any site is called micro-satellite stable (microsatellite stable, MSS).
In the NCCN Guidelines about colorectal cancer examination in 2011, MSI has become primary test item.1. MSI detects the examination that can be used for HNPCC.Surpass 90% HNPCC specimens performance MSI, the important symbol thing that MSI has become in HNPCC; When highly suspecting HNPCC, need to detect MSI.2. the colorectal cancer patients prognosis bona's of MSI-H mark.MSI-H colorectal cancer patients is longer lifetime, and less recurrence, is considered to one of sign of good prognosis.3. MSI detects the auxiliary curative effect prediction that can be used for fluorouracil drug.ASCO meeting coverage in 2009, in accepting the II phase and III phase colorectal cancer patients of 5-FU adjuvant chemotherapy, MSI is Prognostic Factors independently.The microsatellite instability of prior art generally can detect by following 2 kinds of methods:
1) PCR method: selected special primer, with self healthy tissues, compare, in vitro microsatellite locus is carried out to pcr amplification, the product after amplification is through polyacrylamide gel electrophoresis, through various indicating systems (radioautograph, silver dye etc.), analyzes the change that has or not mobility.
2) gene test, directly checks order to relevant DNA.But expensive because carrying out gene test, required test conditions is higher, not yet universal in clinical application.
PCR method has become at present conventional detection method and has been proved to be the most effective primary dcreening operation means.But regular-PCR is in charge of five micro-satellite genes of detection, and product is done the method for gel electrophoresis, has complex operation, the many defects of high in cost of production.
Chinese patent 201110152226.X discloses a kind of composite amplification system and detection kit of tumour cell microsatellite instability state, chosen 8 accurate monomorphism mononucleotides repetition sites (NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26, MONO-27, CAT-25) MSI has been carried out to the detection of PCR multiplex amplification, for effectively distinguishing and adopted 3 kinds of fluorescent markers.And carry out multiple fluorescent mark, at aspects such as cost control and operability, there is many deficiencies, add sex determination site for preventing that effect that experimental implementation person obscures sample aspect from being also very limited.
In view of the foregoing, provide a kind of improvement, easily, highly sensitive MSI testing product is imperative.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of have specific nucleotide sequence general-distinctive embedment combination of primers, can effectively weaken competition and interfering factors between primer, detected result accuracy and highly sensitive colorectal cancer microsatellite instability amplification system and detection kit thereof, this amplification system can be realized and in a PCR system, adopt 5 microsatellite locus of single fluorescent mark one-time detection.
The present invention is that a kind of technical scheme that solves the problems of the technologies described above proposition is: a kind of colorectal cancer microsatellite instability amplification system, comprises the mixture of general-distinctive embedment primer pair and universal primer;
Described general-distinctive embedment primer pair is respectively for microsatellite locus BAT25, BAT26, D2S123, D5S346 and D17S250; Described general-forward primer in distinctive embedment primer pair is Up-BAT25, Up-BAT26, Up-D2S123, Up-D5S346 and Up-D17S250, corresponding reverse primer is BAT25-R, BAT26-R, D2S123-R, D5S346-R and D17S250-R;
Described universal primer is FluD5-Up, described general-each forward primer 5 ' end in distinctive embedment primer pair is provided with the sequence of FluD5-Up, described FluD5-Up is fluorescent dye primer;
Described general-nucleotide sequence of distinctive embedment primer pair and universal primer is as follows:
Prime Name Prime Sequences（5’-3’）
Up-BAT25 GCCGTCGAACTGTCACCTCGGCTTTCCTCGCCTCCAAG
BAT25-R CACTTCAAAATGACATTCTGCA
Up-BAT26 GTCGAACTGTCACCTCGGACAGTTTGAACTGACTAC
BAT26-R CGCTAGTTATCTAATCCAAG
Up-D2S123 GTCGAACTGTCACCTCCATTGCTGGAAGTTCTGGCC
D2S123-R GACTAACCGTCCCATAGGTGG
Up-D5S346 GTCGAACTGTCACCTCGGCATATGAATACCAGGATAGC
D5S346-R GCAGAGTATCAATCTTGTACC
Up-D17S250 TCGAACTGTCACCTCGATTTACTGTGGATTGGTAAGTC
D17S250-R CAGGCATGAGCCACTCAGCTGGC
FluD5-Up GCCGTCGAACTGTCACCTC。
Technique scheme preferably a kind of: the concentration of above-mentioned primers F luD5-Up is 0.1 μ M～0.25 μ M.
A kind of technique scheme further preferably: the ratio of the concentration of the concentration of above-mentioned primer Up-BAT25 and primers F luD5-Up is 1: 25 to 1: 15; The ratio of the concentration of described primer Up-BAT26, Up-D2S123 and Up-D5S346 and the concentration of primers F luD5-Up is 1: 50 to 1: 30; The ratio of the concentration of the concentration of described primer Up-D17S250 and primers F luD5-Up is 1: 5 to 1: 3; The ratio of the concentration of described primer BAT25-R, BAT26-R, D2S123-R, D5S346-R and D17S250-R and the concentration of primers F luD5-Up is 10: 9 to 5: 3.
A kind of technique scheme further preferably: the concentration of above-mentioned primers F luD5-Up is 0.2 μ M, the concentration of primer Up-BAT25 0.01 μ M, Up-BAT26, Up-D2S123 and Up-D5S346 is all 0.005 μ M, the concentration of primer Up-D17S25 0 is 0.05 μ M, and the concentration of primer BAT25-R, BAT26-R, D2S123-R, D5S346-R and D17S250-R is all 0.25 μ M.
The fluorescent mark of above-mentioned FluD5-Up is Cy5, Cy3 or FAM.Preferably the fluorescent mark of FluD5-Up is Cy5.
The cumulative volume of above-mentioned amplification system is 10 μ l～20 μ l.
Above-mentioned amplification system also comprises the Mg of PCR damping fluid, 1.5mM～2mM
2+, the warm start enzyme of dNTP, 0.4U～0.6U of 0.2 mM and the DNA profiling of 10 ng～200ng.
The present invention is that the another kind of technical scheme that solves the problems of the technologies described above proposition is: a kind of colorectal cancer microsatellite instability detection kit that adopts above-mentioned amplification system.
Positively effect of the present invention is:
1) colorectal cancer microsatellite instability amplification system of the present invention is blended in one by microsatellite marker, Multiplex fluorescent PCR and capillary electrophoresis technique, overcome prior art regular-PCR and can only in a reaction system, detect the deficiency of a micro-satellite gene locus, realized and in a PCR system, detected 5 MSI gene locuss simultaneously, save cost, improved efficiency.Due to adopted specific general-distinctive embedment primer, and apply abiotic source property universal primer and do single fluorescent mark, avoid common multiplex PCR need carry out primer multiple fluorescently-labeled loaded down with trivial details, both weakened competition and interfering factors between primer, saved again expense.
2) multiple PCR products obtains product sheet segment data, photoluminescence peak and collection of illustrative plates after capillary electrophoresis, comparison of tumor tissue and control product clip size, mark fluorescent power and peak type change again, carry out microsatellite instability judgement, forward primer in general in amplification system of the present invention-distinctive embedment primer pair and the concentration of reverse primer can directly affect the result of multiplex PCR, particularly important.Amplification system of the present invention is when preparation, make forward primer Up-BAT25, Up-BAT26, Up-D2S123, Up-D5S346, the concentration of Up-D17S250 is lower than universal primer FluD5-Up concentration, reverse primer BAT25-R, BAT26-R, D2S123-R, D5S346-R, the concentration of D17S250-R is higher than the concentration of FluD5-Up, and the concentration of each primer is controlled in suitable scope, especially the concentration of forward primer Up-BAT25 and Up-D17S250 has been carried out to strict control, thereby competition and interfering factors between primer have more effectively been weakened, make the result of multiplex PCR more reliable.
3) the capillary electrophoresis fragment analysis technology of the fragment analysis of test kit of the present invention application is different from conventional gel electrophoretic analysis pattern, makes PCR interpretation of result more directly perceived, succinct, reliable, is easy to identification judgement, is more conducive to normalizing operation.
4) simple in structure, the simple operation of the present invention, working conditions medelling, be convenient to large-scale promotion application.
Accompanying drawing explanation
Fig. 1 is the multi-PRC reaction principle schematic of detection kit of the present invention;
Fig. 2 is the somatotype collection of illustrative plates that the detection kit of embodiment 1 detects the blood sample of sample 1;
Fig. 3 is the somatotype collection of illustrative plates that the detection kit of embodiment 1 detects the tumor tissues DNA of sample 1;
Fig. 4 is that the detection kit of embodiment 1 detects the blood sample of sample 1 and the somatotype collection of illustrative plates of tumor tissues DNA stack;
Fig. 5 is the somatotype collection of illustrative plates that the detection kit of embodiment 1 detects the blood sample of sample 2;
Fig. 6 is the somatotype collection of illustrative plates that the detection kit of embodiment 1 detects the tumor tissues DNA of sample 2;
Fig. 7 is that the detection kit of embodiment 1 detects the blood sample of sample 2 and the somatotype collection of illustrative plates of tumor tissues DNA stack.
Embodiment
Embodiment 1
The colorectal cancer microsatellite instability detection kit of the present embodiment is at least comprised of six test tubes that corresponding reagent is housed that are fixed in paper package box, and six test tubes are respectively amplification buffer test tube, Mg
2+test tube, dNTP test tube, warm start enzyme test tube, primer mixture test tube and ultrapure water test tube.The order that the arrangement of above-mentioned test tube in packing box placed does not have special requirement, only will be clear that sign.
The developing principle of the detection kit of the present embodiment and using method are:
A.MSI gene locus design of primers, synthetic and screening:
According to Bethesda standard microsatellite site, comprise BAT25, BAT26, D2S123, D5S346, five genes of D17S250, design its polymorphic micro-satellite markers primer.
Design primer is used Oligo7.0 software to carry out performance evaluation, first make each primer Tm value reach the numerical value interval that the temperature difference is not more than 2 ℃, secondly between primer, do not produce any secondary structure, finally apply BLAST determine primer not with other genome generation cross reaction.Designing altogether 15 pairs of primers screens.
Synthetic above-mentioned 15 pairs of primers, screen with colorectal cancer person's DNA sample:
Use DNA cleavage liquid and Proteinase K to extract 2 routine persons' to be tested tumor tissues and blood sample DNA, obtaining concentration of specimens interval is 10 ng/ μ l～200ng/ μ l, and high density sample is diluted with water to 100ng/ μ l.
With above-mentioned 2 routine DNA samples to be measured, first carry out single amplification screening, carry out multiplex amplification again and filter out 5 pairs of primers as PCR system combinations primer, forward primer (STR-F) is called after Up-BAT25, Up-BAT26, Up-D2S123, Up-D5S346 and Up-D17S250 respectively; Corresponding reverse primer (STR-R) is called after BAT25-R, BAT26-R, D2S123-R, D5S346-R and D17S250-R respectively.
Design 3 universal primers, make its sequence not with above-mentioned ten Auele Specific Primer generation secondary structures, and its Tm value is a little less than Auele Specific Primer Tm value, so that Auele Specific Primer first fully amplification in multiplex PCR system.
Universal primer 5 ' end is done fluorescein-labelled, uses respectively FAM, CY3 or CY5 reporter group to mark, result all can, this universal primer called after FluD5-Up.
B. carry out the structure of MSI multiplex PCR-CE system:
The PCR reaction system that the detection kit of the present embodiment adopts is multi-PRC reaction system, and the 5 pairs of micro-satellite gene locus special primers and fluorescent mark universal primer are placed in same PCR pipe and carry out pcr amplification.Adopt 10 μ l～20 μ l reaction systems all can better carry out composite amplification.After considering cost factor and experiment is repeatedly adjusted and optimized through PCR, preferred multi-PRC reaction system cumulative volume is 10 μ l, comprises the Mg of 10ng～200ng genomic dna, PCR damping fluid, 2mM
2+, 0.2mM the warm start enzyme of dNTP, 0.5U, and primer BAT25-R, BAT26-R, D2S123-R, D5S346-R, each 0.25 μ M of D17S250-R, FluD5-Up 0.2 μ M, Up-BAT25 0.01 μ M, Up-BAT26, Up-D2S123, Up-D5S346 each 0.005 μ M, Up D17S250 0.05 μ M.
STR-F concentration in general-distinctive embedment primer pair is number/mono-of the concentration of universal primer FluD5-Up.General-distinctive embedment primer pair (STR-F and STR-R) first carries out the amplification of 5～10 circulations under the effect of warm start enzyme to MSI gene, produce 5 ' end with the complementary fragment of Up; The product with Up that FluD5-Up and STR-R primer obtain pcr amplification under the effect of warm start enzyme afterwards carries out the amplification of follow-up 35～40 circulations again, produces 5 ' end with the complementary MSI gene fragment of Cy5 mark.The principle of above-mentioned PCR reaction as shown in Figure 1.
The preferred PCR reaction conditions of detection kit of the present embodiment is: 95 ℃ of preheatings 5 minutes; Then 94 ℃ of sex change are 10 seconds, 54 ℃ of annealing 10 seconds, and 72 ℃ are extended 30 seconds, totally 40 circulations; Last 72 ℃ are extended 5 minutes, 4 ℃ of insulations.
Through the PCR of above-mentioned steps gained product, carry out capillary electrophoresis (capillary electrophoresis, CE).Utilize multiple fluorescence PCR to detect micro-satellite in conjunction with capillary electrophoresis technique, there is the advantages such as efficient, stable, highly sensitive, analytical results is reliable.Each sample CE system cumulative volume is 20 μ l, comprises above-mentioned PCR product 1 μ l～5ul, DNA-Size reference liquid 0.2 μ l～0.5 μ l, and Sample Loading Solution and paraffin oil, and electrophoresis gained fluorescence pattern carries out fragment analysis.
C. colorectal cancer cell MSI gene fragment analysis judgement:
According to gene fragment size and the comparison of photoluminescence peak peak shape in capillary electrophoresis collection of illustrative plates, if tumor tissues occurs a certain gene band displacement or obtain extra banding pattern compared with whole blood sample being judged as a certain genic instability change.The standard of judgement adopts international cancer association to recommend the MSl classification guidance program of formulating: detect and analyze in 5 microsatellite locus of MSI state, 2 or 2 unstable height microsatellite instabilities (MSI-H) that are judged to be in above site, 1 unstable low microsatellite instability (MSI-L) that is judged to be in site, without the unstable micro-satellite stable (MSS) that is judged to be in site.
The use step of the detection kit of the present embodiment is: the DNA extraction of first carrying out colorectal cancer person's tumor tissues and contrast; Adopt again multiplex amplification system increase and detect; Finally carry out the analysis judgement of multiple product.
Below to adopt the detection kit of the present embodiment two routine colorectal cancer persons to be detected to the concrete performance of MSI state:
1, whole blood and tumor tissues paraffin section DNA extraction:
Extract respectively whole blood DNA and the tumor tissues DNA of two routine samples to be tested, called after sample 1 and 2.
These 1 or 2 whole blood 400 μ l of sampling, add 1ml distilled water, mix latter centrifugal 5 minutes, discard upper strata liquid 1ml, add 400 μ l lysates and 20 μ l Proteinase Ks, 70 degree metal baths 10 minutes, cross centrifugal column, 500 μ l scavenging solutions successively clean after twice, add the centrifugal wash-out of 50 μ l elutriant, survey sample DNA concentration value.Adjust DNA concentration to 50ng/ μ l.
5 of this tumor tissues paraffin sections of 1 or 2 of sampling, scraping blade enters 1.5ml centrifuge tube, add the dewaxing of 1ml dimethylbenzene, 1ml ethanol cleans after twice, adds 400 μ l lysates and 20 μ l Proteinase Ks, 60 degree metal baths 16 hours, cross centrifugal column, 500 μ l scavenging solutions successively clean after twice, add the centrifugal wash-out of 50 μ l elutriant, survey sample DNA concentration value, adjust DNA concentration to 50ng/ μ l.
2, multiplex PCR-CE method detects MSI, and concrete operation step is as follows:
A. in each reaction system, add 10 * PCR damping fluid, 1 μ l, 100mM Mg
2+0.2 μ l, 2.5mM dNTP 0.8 μ l, 5U warm start enzyme 0.1 μ l, primer mixture (comprise 25uM BAT25-R, BAT26-R, D2S123-R, D5S346-R, D17S250-R each 0.1 μ l, 25 μ M FluD5-Up 0.08 μ l, 25 μ M Up-BAT25 0.004 μ l, 25 μ M Up-BAT26, Up-D2S123, Up-D5S346-R each 0.002 μ l, 25 μ M Up-D17S250 0.02 μ l), the DNA profiling 2 μ l that above-mentioned extracting obtains, moisturizing to 10 μ l.
The sequence following (5 '-3 ') of the primer that adopts:
BAT25 primer
Up-BAT25：GCCGTCGAACTGTCACCTCGGCTTTCCTCGCCTCCAAG
BAT25-R：CACTTCAAAATGACATTCTGCA
BAT26 primer
Up-BAT26：GCCGTCGAACTGTCACCTCGGACAGTTTGAACTGACTAC
BAT26-R：CGCTAGTTATCTAATCCAAG
D2S123 primer
Up-D2S123：GCCGTCGAACTGTCACCTCCATTGCTGGAAGTTCTGGCC
D2S123-R：GACTAACCGTCCCATAGGTGG
D5S346 primer
Up-D5S346：GCCGTCGAACTGTCACCTCGGCATATGAATACCAGGATAGC
D5S346-R：GCAGAGTATCAATCTTGTACC
D17S250 primer
Up-D17S250：GCCGTCGAACTGTCACCTCGATTTACTGTGGATTGGTAAGTC
D17S250-R：CAGGCATGAGCCACTCAGCTGGC
The sequence of universal primer FluD5-Up is: GCCGTCGAACTGTCACCTC.
5 ' the end of universal primer FluD5-Up is with fluorescent mark.
B. PCR reaction tubes is placed on amplification instrument, the PCR response procedures of operation is: 95 ℃ of preheatings 5 minutes; Then 94 ℃ of sex change are 10 seconds, 54 ℃ of annealing 10 seconds, and 72 ℃ are extended 30 seconds, totally 40 circulations; Last 72 ℃ are extended 5 minutes.4 ℃ of preservations.
C. gained PCR product is placed on Beckman GenomelabTM GeXP genetic analyzer, by instrument operation instruction, carry out capillary electrophoresis, each CE system cumulative volume is 20 μ l, comprises above-mentioned PCR product 1 μ l, DNA-Size reference liquid 0.5 μ l, Sample Loading Solution 18.5 μ l.CE gained fluorescence pattern carries out gene fragment analysis.
3, Sporadic Colorectal Carcinoma MSI gene fragment is analyzed:
The whole blood of above-mentioned sample 1 or 2 and tumor tissues sample CE collection of illustrative plates are carried out to gene fragment analysis, according to the gene fragment size indicating in collection of illustrative plates and photoluminescence peak peak shape, do relatively synchronous, as shown in Figures 2 to 7, X-coordinate is gene fragment base numerical value, ordinate zou is fluorescence numerical value, X-coordinate gene fragment from left to right is sequentially followed successively by for microsatellite locus D2S123, BAT26, BAT-25, D17S250, the amplified fragments of D5S346, wherein Fig. 2 and Fig. 5 are whole blood sample gene fragment analysis chart, Fig. 3 and Fig. 6 are tumor tissues sample chips piecewise analysis figure, Fig. 4 and Fig. 7 are the comparison diagram after superposeing.All there is not the displacement phenomenon of gene fragment in sample 1 wherein, therefore be judged to be micro-satellite stable (MSS); Sample 2 tumor tissues sample D17S250 compare blood sample and all occur band displacement phenomenon with D5S346 gene fragment, so detected person is height microsatellite instability (MSI-H).
To the blood sample of sample 1 and 2 and tumor tissues sample, adopt respectively: (1) BAT25 primer+universal primer; (2) BAT26 primer+universal primer; (3) D2S123 primer+universal primer; (4) D5S346 primer+universal primer; (5) D17S250 primer+universal primer, carries out adopting capillary electrophoresis analysis after substance pcr amplification, and result of determination is consistent with above-mentioned multiplex PCR result.
Embodiment 2
The rest part of the colorectal cancer microsatellite instability detection kit of the present embodiment is identical with embodiment 1, and difference is: the forward primer in general-distinctive embedment primer pair is different with the concentration of reverse primer.
Embodiment 3
The rest part of the colorectal cancer microsatellite instability detection kit of the present embodiment is identical with embodiment 1, and difference is: the forward primer in general-distinctive embedment primer pair is different with the concentration of reverse primer.
Comparative example 1 to 3
The rest part of the detection kit of comparative example 1 to 3 is identical with embodiment 1, and difference is: the forward primer in general-distinctive embedment primer pair is different with the concentration of reverse primer.
Forward primer in the detection kit of embodiment 1 to 3 and comparative example 1 to 3 and the concentration of reverse primer are as shown in table 1:
Table 1 primer concentration table
The detected result of embodiment 2 and 3 detection kit is consistent with the detected result of embodiment 1, the detected result of the detection kit of comparative example 1 to 3 and the detected result of embodiment 1 be deviation to some extent, as shown in table 2, hence one can see that, amplification system is when preparation, make the concentration of forward primer Up-BAT25, Up-BAT26, Up-D2S123, Up-D5S346, Up-D17S250 must be lower than universal primer FluD5-Up concentration, otherwise a little less than can making fluorescent signal too; The concentration of reverse primer BAT25-R, BAT26-R, D2S123-R, D5S346-R, D17S250-R must be higher than the concentration of FluD5-Up, so that PCR sufficient reacting; And the concentration of each primer should be controlled in suitable scope, especially to the concentration of forward primer Up-BAT25 and Up-D17S250, must strictly control, otherwise can cause the interference between primer, in the situation that some tissue samples concentration is on the low side, the amplification efficiency that more easily makes some site is too low and cannot identification, causes judging MSI result.
Embodiment 1 to 3 makes by optimizing primer concentration to have reached balance between the peak type of each site amplified fragments.If different tissues gene amplification efficiency is suitable, electrophorogram can be superimposed to analyze, as Fig. 4 and Fig. 7; If wherein a certain amplification efficiency of organizing is lower, can the some multiples of corresponding amplification to analyze, it is standard that magnification be take the clear and legible knowledge of object fragment.
Table 2 detected result table
Obviously, above-described embodiment is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And these belong to apparent variation that spirit of the present invention extended out or change still among protection scope of the present invention.
Claims (8)
1. a colorectal cancer microsatellite instability amplification system, is characterized in that: the mixture that comprises general-distinctive embedment primer pair and universal primer;
Described general-distinctive embedment primer pair is respectively for microsatellite locus BAT25, BAT26, D2S123, D5S346 and D17S250; Described general-forward primer in distinctive embedment primer pair is Up-BAT25, Up-BAT26, Up-D2S123, Up-D5S346 and Up-D17S250, corresponding reverse primer is BAT25-R, BAT26-R, D2S123-R, D5S346-R and D17S250-R;
Described universal primer is FluD5-Up, described general-each forward primer 5 ' end in distinctive embedment primer pair is provided with the sequence of FluD5-Up, described FluD5-Up is fluorescent dye primer;
Described general-nucleotide sequence of distinctive embedment primer pair and universal primer is as follows:
Prime Name Prime Sequences（5’-3’）
Up-BAT25 GCCGTCGAACTGTCACCTCGGCTTTCCTCGCCTCCAAG
BAT25-R CACTTCAAAATGACATTCTGCA
Up-BAT26 GTCGAACTGTCACCTCGGACAGTTTGAACTGACTAC
BAT26-R CGCTAGTTATCTAATCCAAG
Up-D2S123 GTCGAACTGTCACCTCCATTGCTGGAAGTTCTGGCC
D2S123-R GACTAACCGTCCCATAGGTGG
Up-D5S346 GTCGAACTGTCACCTCGGCATATGAATACCAGGATAGC
D5S346-R GCAGAGTATCAATCTTGTACC
Up-D17S250 TCGAACTGTCACCTCGATTTACTGTGGATTGGTAAGTC
D17S250-R CAGGCATGAGCCACTCAGCTGGC
FluD5-Up GCCGTCGAACTGTCACCTC。
2. colorectal cancer microsatellite instability amplification system according to claim 1, is characterized in that: the concentration of described primers F luD5-Up is 0.1 μ M～0.25 μ M.
3. colorectal cancer microsatellite instability amplification system according to claim 2, is characterized in that: the ratio of the concentration of the concentration of described primer Up-BAT25 and primers F luD5-Up is 1: 25 to 1: 15; The ratio of the concentration of described primer Up-BAT26, Up-D2S123 and Up-D5S346 and the concentration of primers F luD5-Up is 1: 50 to 1: 30; The ratio of the concentration of the concentration of described primer Up-D17S250 and primers F luD5-Up is 1: 5 to 1: 3; The ratio of the concentration of described primer BAT25-R, BAT26-R, D2S123-R, D5S346-R and D17S250-R and the concentration of primers F luD5-Up is 10: 9 to 5: 3.
4. colorectal cancer microsatellite instability amplification system according to claim 3, it is characterized in that: the concentration of described primers F luD5-Up is 0.2 μ M, the concentration of primer Up-BAT25 0.01 μ M, Up-BAT26, Up-D2S123 and Up-D5S346 is all 0.005 μ M, the concentration of primer Up-D17S25 0 is 0.05 μ M, and the concentration of primer BAT25-R, BAT26-R, D2S123-R, D5S346-R and D17S250-R is all 0.25 μ M.
5. according to the colorectal cancer microsatellite instability amplification system one of claim 1 to 4 Suo Shu, it is characterized in that: the fluorescent mark of described FluD5-Up is Cy5, Cy3 or FAM.
6. according to the colorectal cancer microsatellite instability amplification system one of claim 1 to 4 Suo Shu, it is characterized in that: the cumulative volume of described amplification system is 10 μ l～20 μ l.
7. according to the colorectal cancer microsatellite instability amplification system one of claim 1 to 4 Suo Shu, it is characterized in that: the Mg that also comprises PCR damping fluid, 1.5mM～2mM
2+, the warm start enzyme of dNTP, 0.4U～0.6U of 0.2 mM and the DNA profiling of 10 ng～200ng.
8. a colorectal cancer microsatellite instability detection kit that adopts amplification system as claimed in claim 1.
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